- Email: [email protected]
Carthami flos regulates gastrointestinal motility functions
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Original Article
Carthami flos regulates gastrointestinal motility functions Iksung Kim, Jinsoo Bae, Byung Joo Kim ∗ Division of Longevity and Biofunctional Medicine, Pusan National University School of Korean Medicine, Yangsan 50612, Republic of Korea
a r t i c l e
i n f o
a b s t r a c t
Article history:
Background: Gastrointestinal (GI) symptoms are common in the general population. This
Received 31 July 2017
investigation studied the effects of Carthami flos (CF), a natural product, on GI motility.
Received in revised form
Methods: We checked the intestinal transit rates (ITRs) or gastric emptying in normal and
14 August 2017
in GI-motility-dysfunction (GMD) mice in vivo. The GMD mice were made by acetic acid or
Accepted 27 August 2017
streptozotocin.
Available online xxx
Results: Both ITRs and gastric emptying were increased by CF (0.0025–0.25 g/kg) dose dependently. Also, in the GMD mice models, acetic-acid-induced peritoneal irritation, and
Keywords:
streptozotocin-induced diabetic mice, the ITRs were decreased compared to normal mice,
Carthami flos
and these decreases were inhibited by CF.
Gastric emptying
Conclusion: These results suggest that CF is one of the good candidates for the development
Gastrointestinal motility
of a prokinetic agent that may regulate GI-motility functions.
GI-motility dysfunction
© 2017 Korea Institute of Oriental Medicine. Published by Elsevier. This is an open access
Intestinal transit rate
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1.
Introduction
Gastrointestinal (GI) motility disorders, which are common in the general population, lead to a substantial decrease in quality of life. Frequent GI-motility-related symptoms include constipation, abdominal pain, bloating, vomiting, diarrhea, and incomplete evacuation.1 These symptoms may be linked to motility disturbances caused by either prolonged/accelerated GI transit or peristaltic dyscoordination.2 Traditional Chinese medicine has a crucial role in the treatment of various diseases. Carthami flos (CF, also known as Carthamus tinctorius L.) is an annual safflower belonging to
the family Compositae. The major constituents of CF are glycerides, linoleic acid, oleic acid, and so on.3 CF has been used for centuries in Asia to treat cervical disk herniation,4 shoulder pain,5 degenerative knee arthritis,6 urological diseases,7 and lung inflammation.8 In addition, CF is used for blood stasis caused by sprains or accidents, and to treat both baldness and pain caused by kidney deficiency.9 In the small intestinal interstitial cells of Cajal (ICC) of mice, CF depolarized pacemaker potentials.10 However, few animal studies have assessed the effects of CF extract on GI-motility functions. Therefore, this investigation studied the effects of CF extract on GI motor functions by measuring the intestinal transit rates (ITRs) and gastric emptying (GE) in an in vivo mouse model.
∗
Corresponding author. E-mail address: [email protected] (B.J. Kim). http://dx.doi.org/10.1016/j.imr.2017.08.005 2213-4220/© 2017 Korea Institute of Oriental Medicine. Published by Elsevier. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Kim I, et al. Carthami flos regulates gastrointestinal motility functions. Integr Med Res (2017), http://dx.doi.org/10.1016/j.imr.2017.08.005
IMR-270; No. of Pages 5
ARTICLE IN PRESS
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2.
Methods
2.1.
Ethics
Animal care and experiments were conducted in accordance with the guidelines issued by the ethics committee of Pusan National University (Busan, Korea; approval number PNU2016-1370) and those issued by the National Institute of Health’s Guide for the Care and Use of Laboratory Animals.
2.2.
CF preparation
The powder of an aqueous extract of CF (C. tinctorius L.; CW04082) was obtained from the plant-extract bank at the Korea Research Institute of Bioscience & Biotechnology (Daejeon, Republic of Korea). The powder was immersed in distilled water (DW) and extracted for 2.5 hours. After that, it was evaporated under reduced pressure using a DW-290 (Daewoong, Seoul, Republic of Korea) at 100 ◦ C. The extract was then lyophilized using a Clean-vac 12 dryer (Biotron Electronics Corporation, Calgary, Alberta, Canada) for 24 hours. Next, CF was dissolved in DW at a concentration of 20 mg/mL, and stored at 4 ◦ C as a stock solution.
2.3.
Animals
To check the in vivo effects of the CF extract on the GI tract, male Institute of Cancer Research (ICR) mice were used (Samtako BioKorea Co., Ltd., Osan, Republic of Korea). The animals were maintained under controlled conditions (weighing 22–24 g, 21 ± 2 ◦ C, relative humidity 51 ± 6%, lights on 7:00 am to 7:00 pm). The animals were deprived of food for 24 hours prior to the experiments.
2.4.
Measurement of ITR using Evans blue
After administering CF extract to normal ICR mice intragastrically, 5% Evans-blue solution was administered (0.1 mL/kg) through an orogastric tube. The animals were sacrificed after Evans-blue administration, and we checked the dye distance in the intestine from the pylorus to its most distal point.
2.6.
After treatment with CF extract, a 0.05% phenol-red solution was administered. Twenty minutes later, the mice were sacrificed and stomachs were removed. The removed stomachs were cut into several pieces in 0.01N NaOH (5 mL) and treated with 20% trichloroacetic acid (0.2 mL). Next, the mixtures were centrifuged for 10 minutes (1050×g), and after that, the 0.05 mL supernatants were added to 0.2 mL NaOH (0.5N). The absorbances were subsequently measured using a spectrometer at 560 nm. The GE value (%) was calculated by 100–(A/B) × 100, where A was the test stomach absorbance, and B was the control stomach absorbance.
2.7.
Drugs
The dried immature fruit of Poncirus trifoliata Raf. (PF) was prepared as previously described,14,15 and its prokinetic activities were compared with those of the CF extract. This PF was selected because it is one of the most popular traditional folk medicines in Korea and exerted potent prokinetic activities in animals.16 All drugs were purchased from Sigma-Aldrich.
2.8.
Statistical analysis
The results are expressed as the means ± standard errors. Statistical analysis was performed using Student t test or by analysis of variance followed by Tukey’s multiple-comparison test, as appropriate. The statistical significance was accepted for p < 0.05.
3.
Results
3.1.
CF regulates the ITR in normal mice
The mean ITR (%) in normal mice was 54.2 ± 2.2% (Fig. 1). Treatment with PF (1 g/kg) increased the ITR [72.3 ± 5.0% (p < 0.01)]. Treatment with the CF extract also increased the ITR (%) in a dose-dependent manner, with ITR values at 0.0025 g/kg, 0.025 g/kg, and 0.25 g/kg of 55.4 ± 2.6%, 65.7 ± 2.5% (p < 0.05), and 74.2 ± 2.2% (p < 0.01), respectively, being observed (Fig. 1).
3.2. 2.5.
GE evaluation
CF regulates the ITR in mice with GMD
GI-motility-dysfunction mice model
Two experimental GI-motility-dysfunction (GMD) models were made: a peritoneal-irritation model by injecting 0.6% acetic acid (AA) intraperitoneally at 10 mL/kg,11,12 and a diabetic model by streptozotocin (STZ). For the STZ-induced diabetic model, male ICR mice (aged 4 weeks) were used. To produce diabetes, the mice were administered with STZ (Sigma-Aldrich, St. Louis, MO, USA) solution. Fresh STZ was prepared in citrate buffer (0.1 M/L; pH = 4.0), and administered at 200 mg/kg body weight.13 Two months after STZ injection, blood was withdrawn from a tail vein and blood-glucose concentrations were measured using a ONETOUCH SelectSimple kit (Johnson & Johnson Medical Company, New Brunswick, USA). Diabetes was defined as a blood-glucose level of >16 mM.
To investigate the effects of the CF extract on the GI-motility functions, we used an AA- and an STZ-induced experimental GMD models. As mentioned earlier, the AA model showed a significant inhibition of ITR (%) [29.9 ± 4.5% (p < 0.01 vs. normal); Fig. 2]. However, a significant retardation of this inhibition was observed when the CF extract was administered at 0.0025 g/kg, 0.025 g/kg, or 0.25 g/kg intragastrically [33.4 ± 2.6%, 40.1 ± 1.9%, and 64.5 ± 3.9% (p < 0.01), respectively; Fig. 2]. Also, the STZ-induced diabetic models also showed a significant ITR (%) retardation (42.5 ± 2.6%; Fig. 3), which was also significantly inhibited by the CF extract at 0.0025 g/kg, 0.025 g/kg, or 0.25 g/kg [44.8 ± 1.9% (p < 0.01), 52.6 ± 1.8% (p < 0.01), and 61.1 ± 2.9% (p < 0.01), respectively; Fig. 3]. No abnormal clinical signs or changes were observed in the experimental GMD
Please cite this article in press as: Kim I, et al. Carthami flos regulates gastrointestinal motility functions. Integr Med Res (2017), http://dx.doi.org/10.1016/j.imr.2017.08.005
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70
ITR of Evans blue (%)
70 60 50 40 30 20 10 0 0.0025
PF (1g /kg , i. g.)
CTRL
0.025
30 20 10 Normal CTRL
(1 g/ kg ,
0.0025 0.025
0.25
Carthami flos (g/kg, i.g.)
PF
*
*
CF regulates GE in normal mice
The CF extract (0.0025 g/kg, 0.025 g/kg, and 0.25 g/kg)treated groups showed significantly enhanced GE (%) values when compared to the normal group [GE values at CF extract 0.0025 g/kg, 0.025 g/kg, and 0.25 g/kg were 54.5 ± 1.5%, 55.3 ± 1.2%, and 64.7 ± 1.1% (p < 0.01), respectively; Fig. 4]. The 0.25 g/kg CF extract had effects similar to those of mosapride at 5 mg/kg [67.7 ± 2.2% (p < 0.01)] and domperidone at 5 mg/kg [64.1 ± 1.7% (p < 0.01)] (Fig. 4).
60 50 40 30 20 10
4. Normal CTRL
0.0025 0.025
Discussion
0.25
Carthami flos (g/kg, i.g.)
PF
(1 g/ kg ,
0
i.g .)
ITR of Evans blue (%)
40
Fig. 3 – Effect of aqueous extract of CF on ITR in STZ-induced diabetic mice. Two months after administering STZ, the mice were treated with the CF extract, and then ITRs were determined 30 minutes after Evans-blue administration. Bars represent means ± SEs. *p < 0.01. CF, Carthami flos; CTRL, control; ITR, intestinal transit rate; PF, Poncirus trifoliata Raf.; SE, standard error; STZ, streptozotocin; i.g., intragastric.
3.3. *
50
Carthami flos (g/kg, i.g.)
Fig. 1 – Effect of aqueous extract of CF on ITR (%) in mice. The mice were treated with CF extract, and then ITR were determined 30 minutes after Evans-blue administration. Bars represent means ± SEs. *p < 0.05. † p < 0.01. CF, Carthami flos; CTRL, control; ITR, intestinal transit rate; PF, Poncirus trifoliata Raf.; SE, standard error; i.g., intragastric.
70
*
*
*
60
0
0.25
*
i.g .)
80
ITR of Evans blue (%)
*
†
Fig. 2 – Effect of aqueous extract of CF on ITR in AA-treated mice. After the AA-treated mice were made, the mice were treated with CF extract, and then the ITRs were determined 30 minutes after Evans-blue administration. Bars represent the means ± SEs. *p < 0.01. AA, acetic acid; CF, Carthami flos; CTRL, control; ITR, intestinal transit rate; PF, Poncirus trifoliata Raf.; SE, standard error; i.g., intragastric.
models. These results indicate that the CF extract increased the ITR in the GMD mice.
GI-motility disorders have been diagnosed with increased frequency. Although the initial medical management is the usual course, surgical procedures are increasingly being applied to treat these disorders now.17 GI-motility disorders occur when the gut has lost its ability to coordinate smooth muscle–ICC–neuron regulation by endogenous or exogenous agents.18,19 GI-motility disorders are of clinical importance in many bowel disorders.20 GI-motility-modulating drugs include all compounds that have pharmacological activity of modulating (stimulating or inhibiting) GI motility, which are mainly used for the treatment of functional GI diseases. Moreover, many new GI-motility-modulating drugs are currently under investigation.21 We previously investigated the effects of CF on ICC in the mouse small intestine.10 CF did not show cytotoxic activity and induced antioxidant activity on ICC. Moreover, CF depolarized the pacemaker potentials of ICC in a concentration-dependent manner.10 Therefore, we believe that the GI tract can be a target of CF, and their inter-
Please cite this article in press as: Kim I, et al. Carthami flos regulates gastrointestinal motility functions. Integr Med Res (2017), http://dx.doi.org/10.1016/j.imr.2017.08.005
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*
Gastric Emptying (%)
70
*
*
60 50 40 30 20 10 0
Normal
Mosa.
Dom. 0.0025 0.025
0.25
and preparations. Indeed, the Food and Drug Administration estimates that around 29,000 herbal, vitamins, or supplements are available,27 and approximately 11–43% of patients with GI disorders use alternative or complementary techniques.28–30 Therefore, we believe that CF may be a good gastroprokinetic agent and that future studies to investigate its side effects are warranted. In summary, both the ITR and GE values were dose-dependently increased by the CF extract. Furthermore, the ITRs of GMD mice were significantly lower than those of normal mice, and these inhibitions were significantly inhibited by the CF extract. Taken together, our results suggest that CF may be one of the good candidates for the development of a gastroprokinetic agent.
Carthami flos (g/kg, i.g.)
Fig. 4 – Effect of aqueous extract of CF on GE. After a 24-hour fast, the mice were orally administered with the indicated dosages of CF extract, 5 mg/kg mosapride (a 5-HT4 receptor agonist), 5 mg/kg domperidone (a dopamine receptor antagonist), or DW (control). Bars represent the means ± SEs. *p < 0.01. CF, Carthami flos; CTRL, control; Dom., domperidone; DW, distilled water; GE, gastric emptying; Mosa., mosapride; SE, standard error; 5-HT4 , 5-hydroxytryptamine receptor 4; i.g., intragastric.
Conflicts of interest The authors have no potential conflict of interest to declare.
Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the (NRF) funded by the Ministry of Education (grant number NRF-2014R1A1A2054469).
references action could affect intestinal motility. In addition, many authors have studied the effects of CF. CF has been used to improve blood circulation by lowering the levels of blood lipids,22 and can modulate neutrophilic lung inflammation.8 Additionally, it is known that CF activates Nrf2; inhibits NF-kappaB activation;23 and suppresses lipopolysaccharideinduced expression of interleukin-1 beta, inducible nitric oxide synthase, and cyclooxygenase-2 in RAW 264.7.24 CF also has antioxidant and hepatoprotective effects in rats.25 However, although the considerable use of CF, little is known about its in vivo effects on GI motility. In this study, the CF extract significantly and dose-dependently accelerated the ITR (Fig. 1). In the experimental GMD models, AA mouse, and STZ-induced mouse, the CF extract significantly inhibited GMD-induced retardation (Figs. 2 and 3), and the CF-treated mice had significantly greater GE values, with 0.25 g/kg having effects similar to those of domperidone and mosapride (Fig. 4). CF induced the pacemaking-activity depolarizations of ICC.10 However, CF did not regulate the pacemaking-activity frequency.10 Generally speaking, the pacemaking-activity depolarizations induced the decrease of frequency.26 Unusually, CF-induced pacemaking-activity frequency had no change.10 Therefore, we think that CF increased the GI motility because of not ICCs pacemaking activity frequency but depolarization regulations. CF induced the pacemaking-activity depolarizations of ICC, and this may induce depolarizations of smooth muscle cells. Therefore, CF might induce the increase of GI motility. Korean or Chinese Oriental medicine may be an attractive alternative based on the perception of their “natural” approach and their low risk of side effects in GI disease. There are many commercially available herbal supplements
1. Chang L. Epidemiology and quality of life in functional gastrointestinal disorders. Aliment Pharmacol Ther 2004;20:31–9. 2. Locke GR, Zinsmeister AR, Fett SL, Melton LJ, Talley NJ. Overlap of gastrointestinal symptom complexes in a US community. Neurogastroenterol Motil 2005;17:29–34. 3. Choi YM, Jung DJ, Kim SH, Kim JU, Yook TH. Repeated intramuscular-dose toxicity test of watersoluble Carthami flos (WCF) pharmacopuncture in Sprague–Dawley rats. J Pharmacopunct 2015;18:36–43. 4. Lee GM, Yeom SC, Kim DH, Ryu SW, Kim DJ, Cho NG, et al. A clinical study of Carthami-flos herbal acupuncture treatment on cervical disc herniation patients. Acupuncture 2006;23:21–35. 5. Jeong DH, Ahn HJ, Hwang KS, Yun KB, Kim TW, Moon JH, et al. Clinical study on effect of Carthami-flos herbal acupuncture therapy on shoulder pain. Acupuncture 2002;19:184–92. 6. Hur TY, Yun MY, Cho EH, Lee OJ, Kim KS, Cho NG. Clinical study on effect of Carthami-flos herbal acupuncture therapy. Acupuncture 2002;19:189–200. 7. Miyaoka R, Monga M. Use of traditional Chinese medicine in the management of urinary stone disease. Int Braz J Urol 2009;35:396–405. 8. Kim J, Woo J, Lyu JH, Song HH, Jeong HS, Ha KT, et al. Carthami flos suppresses neutrophilic lung inflammation in mice, for which nuclear factor-erythroid 2-related factor-1 is required. Phytomedicine 2014;21:470–8. 9. Korean Pharmacopuncture Institute. Pharmacopuncturology: principles and clinical applications. Seoul: Elsevier Korea LLC 2012; 159–62. 10. Song HJ, Kim JA, Han SE, Kim HW, Chae H, Kim BJ, et al. Effects of Carthami flos on interstitial cells of Cajal in the gastrointestinal tract. Korean J Orient Physiol Pathol 2011;25:603–7.
Please cite this article in press as: Kim I, et al. Carthami flos regulates gastrointestinal motility functions. Integr Med Res (2017), http://dx.doi.org/10.1016/j.imr.2017.08.005
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11. Ahn TS, Kim DG, Hong NR, Park HS, Kim H, Ha KT, et al. Effects of Schisandra chinensis extract on gastrointestinal motility in mice. J Ethnopharmacol 2015;169:163–9. 12. Lee MC, Ha W, Park J, Kim J, Jung Y, Kim BJ. Effects of Lizhong Tang on gastrointestinal motility in mice. World J Gastroenterol 2016;22:7778–86. 13. Wu YS, Lu HL, Huang X, Liu DH, Meng XM, Guo X, et al. Diabetes-induced loss of gastric ICC accompanied by up-regulation of natriuretic peptide signaling pathways in STZ induced diabetic mice. Peptides 2013;40:104–11. 14. Kim BJ, Kim HW, Lee GS, Choi S, Jun JY, So I, et al. Poncirus trifoliata fruit modulates pacemaker activity in interstitial cells of Cajal from the murine small intestine. J Ethnopharmacol 2013;149:668–75. 15. Kim HJ, Park SY, Kim DG, Park SH, Lee H, Hwang DY, et al. Effects of the roots of Liriope platyphylla Wang Et Tang on gastrointestinal motility function. J Ethnopharmacol 2016;184:144–53. 16. Lyu JH, Lee HT. Effects of dried Citrus unshiu peels on gastrointestinal motility in rodents. Arch Pharm Res 2013;36:641–8. 17. Thompson JS, Langenfeld SJ, Hewlett A, Chiruvella A, Crawford C, Armijo P, et al. Surgical treatment of gastrointestinal motility disorders. Curr Probl Surg 2016;53:503–49. 18. Di Lorenzo C, Youssef NN. Diagnosis and management of intestinal motility disorders. Semin Pediatr Surg 2010;19:50–8. 19. Keller J, Layer P. Intestinal and anorectal motility and functional disorders. Best Pract Res Clin Gastroenterol 2009;23:407–23. 20. Auteri M, Zizzo MG, Serio R. GABA and GABA receptors in the gastrointestinal tract: from motility to inflammation. Pharmacol Res 2015;93:11–21.
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21. Lee OY. Gastrointestinal motility modulating drugs. J Korean Med Assoc 2009;52:920–7. 22. Lin WC, Lai MT, Chen HY, Ho CY, Man KM, Shen JL, et al. Protective effect of Flos carthami extract against ethylene glycol-induced urolithiasis in rats. Urol Res 2012;40:655–61. 23. Jun MS, Ha YM, Kim HS, Jang HJ, Kim YM, Lee YS, et al. Anti-inflammatory action of methanol extract of Carthamus tinctorius involves in heme oxygenase-1 induction. J Ethnopharmacol 2011;133:524–30. 24. Wang CC, Choy CS, Liu YH, Cheah KP, Li JS, Wang JT, et al. Protective effect of dried safflower petal aqueous extract and its main constituent, Carthamus yellow, against lipopolysaccharide-induced inflammation in RAW264.7 macrophages. J Sci Food Agric 2011;91:218–25. 25. Wu S, Yue Y, Tian H, Li Z, Li X, He W, et al. Carthamus red from Carthamus tinctorius L. exerts antioxidant and hepatoprotective effect against CCI(4)-induced liver damage in rats via the Nrf2 pathway. J Ethnopharmacol 2013;148:570–8. 26. Kim BJ, Lee GS, Kim HW. Involvement of transient receptor potential melastatin type 7 channels on Poncirus fructus-induced depolarizations of pacemaking activity in interstitial cells of Cajal from murine small intestine. Integr Med Res 2013;2:62–9. 27. Chambliss LR. Alternative and complementary medicine: an overview. Clin Obstet Gynecol 2001;44:640–52. 28. Smart HL, Mayberry JF, Atkinson M. Alternative medicine consultations and remedies in patients with the irritable bowel syndrome. Gut 1986;27:826–8. 29. Giese LA. A study of alternative health care use for gastrointestinal disorders. Gastroenterol Nurs 2000;23:19–27. 30. Comar KM, Kirby DF. Herbal remedies in gastroenterology. J Clin Gastroenterol 2005;39:457–68.
Please cite this article in press as: Kim I, et al. Carthami flos regulates gastrointestinal motility functions. Integr Med Res (2017), http://dx.doi.org/10.1016/j.imr.2017.08.005
ARTICLE IN PRESS integr med res x x x ( 2 0 1 7 ) xxx–xxx
Available online at www.sciencedirect.com
Integrative Medicine Research journal homepage: www.imr-journal.com
Original Article
Carthami flos regulates gastrointestinal motility functions Iksung Kim, Jinsoo Bae, Byung Joo Kim ∗ Division of Longevity and Biofunctional Medicine, Pusan National University School of Korean Medicine, Yangsan 50612, Republic of Korea
a r t i c l e
i n f o
a b s t r a c t
Article history:
Background: Gastrointestinal (GI) symptoms are common in the general population. This
Received 31 July 2017
investigation studied the effects of Carthami flos (CF), a natural product, on GI motility.
Received in revised form
Methods: We checked the intestinal transit rates (ITRs) or gastric emptying in normal and
14 August 2017
in GI-motility-dysfunction (GMD) mice in vivo. The GMD mice were made by acetic acid or
Accepted 27 August 2017
streptozotocin.
Available online xxx
Results: Both ITRs and gastric emptying were increased by CF (0.0025–0.25 g/kg) dose dependently. Also, in the GMD mice models, acetic-acid-induced peritoneal irritation, and
Keywords:
streptozotocin-induced diabetic mice, the ITRs were decreased compared to normal mice,
Carthami flos
and these decreases were inhibited by CF.
Gastric emptying
Conclusion: These results suggest that CF is one of the good candidates for the development
Gastrointestinal motility
of a prokinetic agent that may regulate GI-motility functions.
GI-motility dysfunction
© 2017 Korea Institute of Oriental Medicine. Published by Elsevier. This is an open access
Intestinal transit rate
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1.
Introduction
Gastrointestinal (GI) motility disorders, which are common in the general population, lead to a substantial decrease in quality of life. Frequent GI-motility-related symptoms include constipation, abdominal pain, bloating, vomiting, diarrhea, and incomplete evacuation.1 These symptoms may be linked to motility disturbances caused by either prolonged/accelerated GI transit or peristaltic dyscoordination.2 Traditional Chinese medicine has a crucial role in the treatment of various diseases. Carthami flos (CF, also known as Carthamus tinctorius L.) is an annual safflower belonging to
the family Compositae. The major constituents of CF are glycerides, linoleic acid, oleic acid, and so on.3 CF has been used for centuries in Asia to treat cervical disk herniation,4 shoulder pain,5 degenerative knee arthritis,6 urological diseases,7 and lung inflammation.8 In addition, CF is used for blood stasis caused by sprains or accidents, and to treat both baldness and pain caused by kidney deficiency.9 In the small intestinal interstitial cells of Cajal (ICC) of mice, CF depolarized pacemaker potentials.10 However, few animal studies have assessed the effects of CF extract on GI-motility functions. Therefore, this investigation studied the effects of CF extract on GI motor functions by measuring the intestinal transit rates (ITRs) and gastric emptying (GE) in an in vivo mouse model.
∗
Corresponding author. E-mail address: [email protected] (B.J. Kim). http://dx.doi.org/10.1016/j.imr.2017.08.005 2213-4220/© 2017 Korea Institute of Oriental Medicine. Published by Elsevier. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Kim I, et al. Carthami flos regulates gastrointestinal motility functions. Integr Med Res (2017), http://dx.doi.org/10.1016/j.imr.2017.08.005
IMR-270; No. of Pages 5
ARTICLE IN PRESS
2
Integr Med Res ( 2 0 1 7 ) xxx–xxx
2.
Methods
2.1.
Ethics
Animal care and experiments were conducted in accordance with the guidelines issued by the ethics committee of Pusan National University (Busan, Korea; approval number PNU2016-1370) and those issued by the National Institute of Health’s Guide for the Care and Use of Laboratory Animals.
2.2.
CF preparation
The powder of an aqueous extract of CF (C. tinctorius L.; CW04082) was obtained from the plant-extract bank at the Korea Research Institute of Bioscience & Biotechnology (Daejeon, Republic of Korea). The powder was immersed in distilled water (DW) and extracted for 2.5 hours. After that, it was evaporated under reduced pressure using a DW-290 (Daewoong, Seoul, Republic of Korea) at 100 ◦ C. The extract was then lyophilized using a Clean-vac 12 dryer (Biotron Electronics Corporation, Calgary, Alberta, Canada) for 24 hours. Next, CF was dissolved in DW at a concentration of 20 mg/mL, and stored at 4 ◦ C as a stock solution.
2.3.
Animals
To check the in vivo effects of the CF extract on the GI tract, male Institute of Cancer Research (ICR) mice were used (Samtako BioKorea Co., Ltd., Osan, Republic of Korea). The animals were maintained under controlled conditions (weighing 22–24 g, 21 ± 2 ◦ C, relative humidity 51 ± 6%, lights on 7:00 am to 7:00 pm). The animals were deprived of food for 24 hours prior to the experiments.
2.4.
Measurement of ITR using Evans blue
After administering CF extract to normal ICR mice intragastrically, 5% Evans-blue solution was administered (0.1 mL/kg) through an orogastric tube. The animals were sacrificed after Evans-blue administration, and we checked the dye distance in the intestine from the pylorus to its most distal point.
2.6.
After treatment with CF extract, a 0.05% phenol-red solution was administered. Twenty minutes later, the mice were sacrificed and stomachs were removed. The removed stomachs were cut into several pieces in 0.01N NaOH (5 mL) and treated with 20% trichloroacetic acid (0.2 mL). Next, the mixtures were centrifuged for 10 minutes (1050×g), and after that, the 0.05 mL supernatants were added to 0.2 mL NaOH (0.5N). The absorbances were subsequently measured using a spectrometer at 560 nm. The GE value (%) was calculated by 100–(A/B) × 100, where A was the test stomach absorbance, and B was the control stomach absorbance.
2.7.
Drugs
The dried immature fruit of Poncirus trifoliata Raf. (PF) was prepared as previously described,14,15 and its prokinetic activities were compared with those of the CF extract. This PF was selected because it is one of the most popular traditional folk medicines in Korea and exerted potent prokinetic activities in animals.16 All drugs were purchased from Sigma-Aldrich.
2.8.
Statistical analysis
The results are expressed as the means ± standard errors. Statistical analysis was performed using Student t test or by analysis of variance followed by Tukey’s multiple-comparison test, as appropriate. The statistical significance was accepted for p < 0.05.
3.
Results
3.1.
CF regulates the ITR in normal mice
The mean ITR (%) in normal mice was 54.2 ± 2.2% (Fig. 1). Treatment with PF (1 g/kg) increased the ITR [72.3 ± 5.0% (p < 0.01)]. Treatment with the CF extract also increased the ITR (%) in a dose-dependent manner, with ITR values at 0.0025 g/kg, 0.025 g/kg, and 0.25 g/kg of 55.4 ± 2.6%, 65.7 ± 2.5% (p < 0.05), and 74.2 ± 2.2% (p < 0.01), respectively, being observed (Fig. 1).
3.2. 2.5.
GE evaluation
CF regulates the ITR in mice with GMD
GI-motility-dysfunction mice model
Two experimental GI-motility-dysfunction (GMD) models were made: a peritoneal-irritation model by injecting 0.6% acetic acid (AA) intraperitoneally at 10 mL/kg,11,12 and a diabetic model by streptozotocin (STZ). For the STZ-induced diabetic model, male ICR mice (aged 4 weeks) were used. To produce diabetes, the mice were administered with STZ (Sigma-Aldrich, St. Louis, MO, USA) solution. Fresh STZ was prepared in citrate buffer (0.1 M/L; pH = 4.0), and administered at 200 mg/kg body weight.13 Two months after STZ injection, blood was withdrawn from a tail vein and blood-glucose concentrations were measured using a ONETOUCH SelectSimple kit (Johnson & Johnson Medical Company, New Brunswick, USA). Diabetes was defined as a blood-glucose level of >16 mM.
To investigate the effects of the CF extract on the GI-motility functions, we used an AA- and an STZ-induced experimental GMD models. As mentioned earlier, the AA model showed a significant inhibition of ITR (%) [29.9 ± 4.5% (p < 0.01 vs. normal); Fig. 2]. However, a significant retardation of this inhibition was observed when the CF extract was administered at 0.0025 g/kg, 0.025 g/kg, or 0.25 g/kg intragastrically [33.4 ± 2.6%, 40.1 ± 1.9%, and 64.5 ± 3.9% (p < 0.01), respectively; Fig. 2]. Also, the STZ-induced diabetic models also showed a significant ITR (%) retardation (42.5 ± 2.6%; Fig. 3), which was also significantly inhibited by the CF extract at 0.0025 g/kg, 0.025 g/kg, or 0.25 g/kg [44.8 ± 1.9% (p < 0.01), 52.6 ± 1.8% (p < 0.01), and 61.1 ± 2.9% (p < 0.01), respectively; Fig. 3]. No abnormal clinical signs or changes were observed in the experimental GMD
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The CF extract (0.0025 g/kg, 0.025 g/kg, and 0.25 g/kg)treated groups showed significantly enhanced GE (%) values when compared to the normal group [GE values at CF extract 0.0025 g/kg, 0.025 g/kg, and 0.25 g/kg were 54.5 ± 1.5%, 55.3 ± 1.2%, and 64.7 ± 1.1% (p < 0.01), respectively; Fig. 4]. The 0.25 g/kg CF extract had effects similar to those of mosapride at 5 mg/kg [67.7 ± 2.2% (p < 0.01)] and domperidone at 5 mg/kg [64.1 ± 1.7% (p < 0.01)] (Fig. 4).
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Fig. 3 – Effect of aqueous extract of CF on ITR in STZ-induced diabetic mice. Two months after administering STZ, the mice were treated with the CF extract, and then ITRs were determined 30 minutes after Evans-blue administration. Bars represent means ± SEs. *p < 0.01. CF, Carthami flos; CTRL, control; ITR, intestinal transit rate; PF, Poncirus trifoliata Raf.; SE, standard error; STZ, streptozotocin; i.g., intragastric.
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Fig. 1 – Effect of aqueous extract of CF on ITR (%) in mice. The mice were treated with CF extract, and then ITR were determined 30 minutes after Evans-blue administration. Bars represent means ± SEs. *p < 0.05. † p < 0.01. CF, Carthami flos; CTRL, control; ITR, intestinal transit rate; PF, Poncirus trifoliata Raf.; SE, standard error; i.g., intragastric.
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Fig. 2 – Effect of aqueous extract of CF on ITR in AA-treated mice. After the AA-treated mice were made, the mice were treated with CF extract, and then the ITRs were determined 30 minutes after Evans-blue administration. Bars represent the means ± SEs. *p < 0.01. AA, acetic acid; CF, Carthami flos; CTRL, control; ITR, intestinal transit rate; PF, Poncirus trifoliata Raf.; SE, standard error; i.g., intragastric.
models. These results indicate that the CF extract increased the ITR in the GMD mice.
GI-motility disorders have been diagnosed with increased frequency. Although the initial medical management is the usual course, surgical procedures are increasingly being applied to treat these disorders now.17 GI-motility disorders occur when the gut has lost its ability to coordinate smooth muscle–ICC–neuron regulation by endogenous or exogenous agents.18,19 GI-motility disorders are of clinical importance in many bowel disorders.20 GI-motility-modulating drugs include all compounds that have pharmacological activity of modulating (stimulating or inhibiting) GI motility, which are mainly used for the treatment of functional GI diseases. Moreover, many new GI-motility-modulating drugs are currently under investigation.21 We previously investigated the effects of CF on ICC in the mouse small intestine.10 CF did not show cytotoxic activity and induced antioxidant activity on ICC. Moreover, CF depolarized the pacemaker potentials of ICC in a concentration-dependent manner.10 Therefore, we believe that the GI tract can be a target of CF, and their inter-
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and preparations. Indeed, the Food and Drug Administration estimates that around 29,000 herbal, vitamins, or supplements are available,27 and approximately 11–43% of patients with GI disorders use alternative or complementary techniques.28–30 Therefore, we believe that CF may be a good gastroprokinetic agent and that future studies to investigate its side effects are warranted. In summary, both the ITR and GE values were dose-dependently increased by the CF extract. Furthermore, the ITRs of GMD mice were significantly lower than those of normal mice, and these inhibitions were significantly inhibited by the CF extract. Taken together, our results suggest that CF may be one of the good candidates for the development of a gastroprokinetic agent.
Carthami flos (g/kg, i.g.)
Fig. 4 – Effect of aqueous extract of CF on GE. After a 24-hour fast, the mice were orally administered with the indicated dosages of CF extract, 5 mg/kg mosapride (a 5-HT4 receptor agonist), 5 mg/kg domperidone (a dopamine receptor antagonist), or DW (control). Bars represent the means ± SEs. *p < 0.01. CF, Carthami flos; CTRL, control; Dom., domperidone; DW, distilled water; GE, gastric emptying; Mosa., mosapride; SE, standard error; 5-HT4 , 5-hydroxytryptamine receptor 4; i.g., intragastric.
Conflicts of interest The authors have no potential conflict of interest to declare.
Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the (NRF) funded by the Ministry of Education (grant number NRF-2014R1A1A2054469).
references action could affect intestinal motility. In addition, many authors have studied the effects of CF. CF has been used to improve blood circulation by lowering the levels of blood lipids,22 and can modulate neutrophilic lung inflammation.8 Additionally, it is known that CF activates Nrf2; inhibits NF-kappaB activation;23 and suppresses lipopolysaccharideinduced expression of interleukin-1 beta, inducible nitric oxide synthase, and cyclooxygenase-2 in RAW 264.7.24 CF also has antioxidant and hepatoprotective effects in rats.25 However, although the considerable use of CF, little is known about its in vivo effects on GI motility. In this study, the CF extract significantly and dose-dependently accelerated the ITR (Fig. 1). In the experimental GMD models, AA mouse, and STZ-induced mouse, the CF extract significantly inhibited GMD-induced retardation (Figs. 2 and 3), and the CF-treated mice had significantly greater GE values, with 0.25 g/kg having effects similar to those of domperidone and mosapride (Fig. 4). CF induced the pacemaking-activity depolarizations of ICC.10 However, CF did not regulate the pacemaking-activity frequency.10 Generally speaking, the pacemaking-activity depolarizations induced the decrease of frequency.26 Unusually, CF-induced pacemaking-activity frequency had no change.10 Therefore, we think that CF increased the GI motility because of not ICCs pacemaking activity frequency but depolarization regulations. CF induced the pacemaking-activity depolarizations of ICC, and this may induce depolarizations of smooth muscle cells. Therefore, CF might induce the increase of GI motility. Korean or Chinese Oriental medicine may be an attractive alternative based on the perception of their “natural” approach and their low risk of side effects in GI disease. There are many commercially available herbal supplements
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Please cite this article in press as: Kim I, et al. Carthami flos regulates gastrointestinal motility functions. Integr Med Res (2017), http://dx.doi.org/10.1016/j.imr.2017.08.005